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CSF1 is a novel p53 target gene whose protein product functions in a feed-forward manner to suppress apoptosis and enhance p53-mediated growth arrest.

Azzam G, Wang X, Bell D, Murphy ME - PLoS ONE (2013)

Bottom Line: This leads to an increase in p53-mediated growth arrest, along with a concomitant decrease in apoptosis.We show that cells expressing CSF1 and CSF1R are indeed radio-resistant, and further, that this effect requires p53.Finally, these data may explain the association of the P72 variant and the CSF1/CSF1R pathway with increased senescence and radio-resistance in some epithelial tumor types.

View Article: PubMed Central - PubMed

Affiliation: Program in Molecular and Cellular Oncogenesis, The Wistar Institute, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
The p53 tumor suppressor gene has a common polymorphism at codon 72 that alters its function. We previously reported that the proline 72 polymorphic variant of p53 (P72) demonstrates increased ability to transactivate a subset of genes, relative to arginine 72 (R72); one of these genes is macrophage colony stimulating factor (CSF1). At present, the mechanism(s) underlying the increased transcriptional activity of P72 toward genes like CSF1 have not been completely elucidated. Additionally, the consequences of increased transcription of genes like CSF1 by the P72 variant to the downstream p53 pathway are unknown. In this report, we address these issues. We show that the CSF1 gene contains a conserved binding site for p53, and interestingly that the P72 variant shows increased ability to bind to this site. Moreover, we show that increased CSF1/CSF1R signaling in P72 cells feeds back on the p53 pathway to enhance p53 phosphorylation, levels, and transactivation of target genes, particularly the cyclin-dependent kinase inhibitor p21 (CDKN1A). This leads to an increase in p53-mediated growth arrest, along with a concomitant decrease in apoptosis. Notably, the CSF1/CSF1R signaling axis is overexpressed in several epithelial cancers, and there is clinical evidence that this pathway plays a role in radio-resistance of some cancers. We show that cells expressing CSF1 and CSF1R are indeed radio-resistant, and further, that this effect requires p53. These combined data are the first to implicate the CSF1/CSF1R pathway in the decision between p53-mediated growth arrest and apoptosis. They are also the first to highlight a cytokine as influential in cell fate determined by p53 in epithelial cells. Finally, these data may explain the association of the P72 variant and the CSF1/CSF1R pathway with increased senescence and radio-resistance in some epithelial tumor types.

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CSF1R signaling promotes cell survival, cell cycle arrest.A. Top panel: Western analysis of lysates from CSF1R overexpressing (CSF1R) or Vector alone (Vector) tet-inducible p53 H1299 cells, untreated (0 hr) or treated with doxycycline (0.75 ug/ml) for the time points indicated (hours, hrs). Cells were analyzed for the levels of p21 (CDKN1A) and GAPDH control. Bottom panel: Flow cytometric analysis of propidium iodide-stained cells from H1299 tetracycline inducible p53 cells stably infected with CSF1R or Vector, untreated or treated with doxycycline (doxy, 0.75 ug/ml) for 24 hours. The percent of cells with 2n DNA content (G1) are shown. Values shown are the average of 3 independent experiments. Error bars mark standard error. B. Flow cytometric analysis of Annexin V-positive cells in H1299 tetracycline inducible p53 cells stably infected with CSF1R or Vector, untreated or treated with doxycycline (doxy, 0.75 ug/ml) and etoposide (50 uM) for 24 hours. Values shown are the average of 3 independent experiments; the p value compares the level of Annexin V positive cells in vector versus CSF1R cells following treatment with doxycycline and etoposide. Error bars mark standard error. C. Colony forming assays were performed with H1299 tet-inducible p53 cells expressing CSF1R (CSF1R) or Vector alone (Vector). Cells were untreated or treated with doxycycline (0.75 ug/ml) 24 hours prior to irradiation with 0, 5, 10, 15 or 20 Gray (Gy) of gamma-irradiation (Gamma-IR). Doxycycline treatments were continued for 8 days; cells were fed every three days. Colonies were then stained with methylene blue and those colonies with greater than 30 cells were counted. Values shown are the average of 3 independent experiments. Error bars mark standard error.
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pone-0074297-g005: CSF1R signaling promotes cell survival, cell cycle arrest.A. Top panel: Western analysis of lysates from CSF1R overexpressing (CSF1R) or Vector alone (Vector) tet-inducible p53 H1299 cells, untreated (0 hr) or treated with doxycycline (0.75 ug/ml) for the time points indicated (hours, hrs). Cells were analyzed for the levels of p21 (CDKN1A) and GAPDH control. Bottom panel: Flow cytometric analysis of propidium iodide-stained cells from H1299 tetracycline inducible p53 cells stably infected with CSF1R or Vector, untreated or treated with doxycycline (doxy, 0.75 ug/ml) for 24 hours. The percent of cells with 2n DNA content (G1) are shown. Values shown are the average of 3 independent experiments. Error bars mark standard error. B. Flow cytometric analysis of Annexin V-positive cells in H1299 tetracycline inducible p53 cells stably infected with CSF1R or Vector, untreated or treated with doxycycline (doxy, 0.75 ug/ml) and etoposide (50 uM) for 24 hours. Values shown are the average of 3 independent experiments; the p value compares the level of Annexin V positive cells in vector versus CSF1R cells following treatment with doxycycline and etoposide. Error bars mark standard error. C. Colony forming assays were performed with H1299 tet-inducible p53 cells expressing CSF1R (CSF1R) or Vector alone (Vector). Cells were untreated or treated with doxycycline (0.75 ug/ml) 24 hours prior to irradiation with 0, 5, 10, 15 or 20 Gray (Gy) of gamma-irradiation (Gamma-IR). Doxycycline treatments were continued for 8 days; cells were fed every three days. Colonies were then stained with methylene blue and those colonies with greater than 30 cells were counted. Values shown are the average of 3 independent experiments. Error bars mark standard error.

Mentions: We next sought to confirm that the increased phosphorylation and stabilization of p53, as well as the increased transactivation of p21, was a direct result of increased signaling by the CSF1/CSF1R pathway to p53. Toward this end, we conducted experiments in H1299 tet-on p53 cells expressing the CSF1R, in the absence or presence of the CSF1R inhibitor II (Calbiochem). Notably, in the presence of the CSF1R inhibitor II, the phosphorylation of p53 at serine 15 is reduced, as is the expression of the p53 target gene p21 (Fig. 4D). These data confirm that the CSF1/CSF1R pathway signals to induce p53 phosphorylation. We next sought to test the hypothesis that the increased p53 activation in the presence of CSF1/CSF1R signaling might affect the cell fate (cell cycle arrest versus apoptosis) determined by p53. Initially, we tested the level of apoptosis in vector compared to CSF1R cells, and performed these experiments in the presence of doxycycline plus etoposide, in order to increase the number of cells undergoing apoptosis. These experiments revealed that CSF1/CSF1R signaling reduces p53-mediated apoptosis, as evident by decreased cleaved lamin A in the presence of an active CSF1/CSF1R pathway (Fig. 4E). To further confirm the impact of CSF1/CSF1R signaling on the p53 growth arrest/cell death decision, we performed flow cytometry of p53-induced cells in vector versus CSF1R-infected cells, and analyzed growth arrest (propidium iodide staining) and apoptosis (Annexin V staining). These studies consistently revealed increased levels of p21, and increased G1 arrest, in CSF1R-expressing cells compared to vector (Fig. 5A, p<0.001), along with decreased apoptosis of cells in the presence of etoposide (Fig. 5B, pā€Š=ā€Š0.001). These combined data indicate that the CSF1/CSF1R pathway feeds forward to p53 to enhance p53 signaling and growth arrest, and to inhibit p53-mediated apoptosis.


CSF1 is a novel p53 target gene whose protein product functions in a feed-forward manner to suppress apoptosis and enhance p53-mediated growth arrest.

Azzam G, Wang X, Bell D, Murphy ME - PLoS ONE (2013)

CSF1R signaling promotes cell survival, cell cycle arrest.A. Top panel: Western analysis of lysates from CSF1R overexpressing (CSF1R) or Vector alone (Vector) tet-inducible p53 H1299 cells, untreated (0 hr) or treated with doxycycline (0.75 ug/ml) for the time points indicated (hours, hrs). Cells were analyzed for the levels of p21 (CDKN1A) and GAPDH control. Bottom panel: Flow cytometric analysis of propidium iodide-stained cells from H1299 tetracycline inducible p53 cells stably infected with CSF1R or Vector, untreated or treated with doxycycline (doxy, 0.75 ug/ml) for 24 hours. The percent of cells with 2n DNA content (G1) are shown. Values shown are the average of 3 independent experiments. Error bars mark standard error. B. Flow cytometric analysis of Annexin V-positive cells in H1299 tetracycline inducible p53 cells stably infected with CSF1R or Vector, untreated or treated with doxycycline (doxy, 0.75 ug/ml) and etoposide (50 uM) for 24 hours. Values shown are the average of 3 independent experiments; the p value compares the level of Annexin V positive cells in vector versus CSF1R cells following treatment with doxycycline and etoposide. Error bars mark standard error. C. Colony forming assays were performed with H1299 tet-inducible p53 cells expressing CSF1R (CSF1R) or Vector alone (Vector). Cells were untreated or treated with doxycycline (0.75 ug/ml) 24 hours prior to irradiation with 0, 5, 10, 15 or 20 Gray (Gy) of gamma-irradiation (Gamma-IR). Doxycycline treatments were continued for 8 days; cells were fed every three days. Colonies were then stained with methylene blue and those colonies with greater than 30 cells were counted. Values shown are the average of 3 independent experiments. Error bars mark standard error.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3760869&req=5

pone-0074297-g005: CSF1R signaling promotes cell survival, cell cycle arrest.A. Top panel: Western analysis of lysates from CSF1R overexpressing (CSF1R) or Vector alone (Vector) tet-inducible p53 H1299 cells, untreated (0 hr) or treated with doxycycline (0.75 ug/ml) for the time points indicated (hours, hrs). Cells were analyzed for the levels of p21 (CDKN1A) and GAPDH control. Bottom panel: Flow cytometric analysis of propidium iodide-stained cells from H1299 tetracycline inducible p53 cells stably infected with CSF1R or Vector, untreated or treated with doxycycline (doxy, 0.75 ug/ml) for 24 hours. The percent of cells with 2n DNA content (G1) are shown. Values shown are the average of 3 independent experiments. Error bars mark standard error. B. Flow cytometric analysis of Annexin V-positive cells in H1299 tetracycline inducible p53 cells stably infected with CSF1R or Vector, untreated or treated with doxycycline (doxy, 0.75 ug/ml) and etoposide (50 uM) for 24 hours. Values shown are the average of 3 independent experiments; the p value compares the level of Annexin V positive cells in vector versus CSF1R cells following treatment with doxycycline and etoposide. Error bars mark standard error. C. Colony forming assays were performed with H1299 tet-inducible p53 cells expressing CSF1R (CSF1R) or Vector alone (Vector). Cells were untreated or treated with doxycycline (0.75 ug/ml) 24 hours prior to irradiation with 0, 5, 10, 15 or 20 Gray (Gy) of gamma-irradiation (Gamma-IR). Doxycycline treatments were continued for 8 days; cells were fed every three days. Colonies were then stained with methylene blue and those colonies with greater than 30 cells were counted. Values shown are the average of 3 independent experiments. Error bars mark standard error.
Mentions: We next sought to confirm that the increased phosphorylation and stabilization of p53, as well as the increased transactivation of p21, was a direct result of increased signaling by the CSF1/CSF1R pathway to p53. Toward this end, we conducted experiments in H1299 tet-on p53 cells expressing the CSF1R, in the absence or presence of the CSF1R inhibitor II (Calbiochem). Notably, in the presence of the CSF1R inhibitor II, the phosphorylation of p53 at serine 15 is reduced, as is the expression of the p53 target gene p21 (Fig. 4D). These data confirm that the CSF1/CSF1R pathway signals to induce p53 phosphorylation. We next sought to test the hypothesis that the increased p53 activation in the presence of CSF1/CSF1R signaling might affect the cell fate (cell cycle arrest versus apoptosis) determined by p53. Initially, we tested the level of apoptosis in vector compared to CSF1R cells, and performed these experiments in the presence of doxycycline plus etoposide, in order to increase the number of cells undergoing apoptosis. These experiments revealed that CSF1/CSF1R signaling reduces p53-mediated apoptosis, as evident by decreased cleaved lamin A in the presence of an active CSF1/CSF1R pathway (Fig. 4E). To further confirm the impact of CSF1/CSF1R signaling on the p53 growth arrest/cell death decision, we performed flow cytometry of p53-induced cells in vector versus CSF1R-infected cells, and analyzed growth arrest (propidium iodide staining) and apoptosis (Annexin V staining). These studies consistently revealed increased levels of p21, and increased G1 arrest, in CSF1R-expressing cells compared to vector (Fig. 5A, p<0.001), along with decreased apoptosis of cells in the presence of etoposide (Fig. 5B, pā€Š=ā€Š0.001). These combined data indicate that the CSF1/CSF1R pathway feeds forward to p53 to enhance p53 signaling and growth arrest, and to inhibit p53-mediated apoptosis.

Bottom Line: This leads to an increase in p53-mediated growth arrest, along with a concomitant decrease in apoptosis.We show that cells expressing CSF1 and CSF1R are indeed radio-resistant, and further, that this effect requires p53.Finally, these data may explain the association of the P72 variant and the CSF1/CSF1R pathway with increased senescence and radio-resistance in some epithelial tumor types.

View Article: PubMed Central - PubMed

Affiliation: Program in Molecular and Cellular Oncogenesis, The Wistar Institute, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
The p53 tumor suppressor gene has a common polymorphism at codon 72 that alters its function. We previously reported that the proline 72 polymorphic variant of p53 (P72) demonstrates increased ability to transactivate a subset of genes, relative to arginine 72 (R72); one of these genes is macrophage colony stimulating factor (CSF1). At present, the mechanism(s) underlying the increased transcriptional activity of P72 toward genes like CSF1 have not been completely elucidated. Additionally, the consequences of increased transcription of genes like CSF1 by the P72 variant to the downstream p53 pathway are unknown. In this report, we address these issues. We show that the CSF1 gene contains a conserved binding site for p53, and interestingly that the P72 variant shows increased ability to bind to this site. Moreover, we show that increased CSF1/CSF1R signaling in P72 cells feeds back on the p53 pathway to enhance p53 phosphorylation, levels, and transactivation of target genes, particularly the cyclin-dependent kinase inhibitor p21 (CDKN1A). This leads to an increase in p53-mediated growth arrest, along with a concomitant decrease in apoptosis. Notably, the CSF1/CSF1R signaling axis is overexpressed in several epithelial cancers, and there is clinical evidence that this pathway plays a role in radio-resistance of some cancers. We show that cells expressing CSF1 and CSF1R are indeed radio-resistant, and further, that this effect requires p53. These combined data are the first to implicate the CSF1/CSF1R pathway in the decision between p53-mediated growth arrest and apoptosis. They are also the first to highlight a cytokine as influential in cell fate determined by p53 in epithelial cells. Finally, these data may explain the association of the P72 variant and the CSF1/CSF1R pathway with increased senescence and radio-resistance in some epithelial tumor types.

Show MeSH
Related in: MedlinePlus